This invention relates to novel oxetanone derivative compounds and processes for producing such derivatives which are useful as lipase inhibitors. Further the invention relates to processes for producing salts and for producing pharmaceutical compositions compounds comprising at least one such oxetanone derivative or salt, as well as methods for using such compounds and compositions for inhibiting lipases. In one aspect the invention relates to lipase inhibitors which include on the same molecule an oxetanone derivative portion capable of inhibiting a lipase and a non-absorbable moiety such a polysaccharide, which are covalently linked or are in the form of a salt. In a preferred aspect of the invention the non-absorbable moiety is lipophilic and will associate with oils or fats. An absorbable oxetanone lipase inhibitor may be rendered non-absorbable by covalent linking it directly or indirectly to a non-absorbable moiety and thereby producing a novel non-absorbable lipase inhibitor.
Some lipase-inhibiting oxetanones and intermediates for making them are well known. See for example, U.S. Pat. Nos. 5,931,463, 4,189,438 and 4,202,824. However, there is a need for improved oxetanones that are have low toxicity and are essentially not absorbable by the digestive system of mammals such as dogs, cats, non-human primates and human primates.
Lipase inhibitors such as esterastin (see U.S. Pat. No. 4,189,438), tetrahydroesterastin (3,5-hydroxy-2hexadeca-7,10-dienoic 1,3-lactone), 3,5-dihydroxy-2-hexylhexadeca-7,10-dienoic 1,3-lactone, 3,5-di-hydroxy-2-hexylhexadecanoic 1,3-lactone, and the like, are well-known as lipase inhibitors and as pancreatic cholesterol esterase inhibitors. However, such lipase inhibitors are, inter alia, also substantially orally active as immunosuppressants (see U.S. Pat. No. 4,189,438 and U.S. Pat. No. 4,202,824), which can be a highly undesired side activity in a normal or immunosuppressed person. Such lipase inhibitor compounds are 3,5 dihydroxy 1,3 lactone derivative compounds, wherein the 5 hydroxyl group may be esterified at the 5 position or is hydrolyzed to the free hydroxyl group.
A popular lipase inhibiting compound which is substantially non-absorbable is known as Orlistat ((2S,3S,5S)-5-[(S)-2-formamido-4-methyl-valeryloxy]-2-hexyl-3-hydroxy-hexadecanoic 1,3 acid lactone, see U.S. Pat. No. 5,643,874). This compound is a steric isomer derivative of tetrahydroesteratin and its 5-hydroxyl group is esterified with a [S-2-formamido-4-methyl-valeryloxy] group. Orlistat has been used to inhibit lipases in the body and thereby prevent the absorption of dietary fat. At a 120 mg dose of Orlistat, taken before consuming a fat-containing meal (or up to one hour after eating such a meal), up to one-third of the fat eaten at a given meal will not be absorbed by the average person and utilized as dietary fat calories. The undigested fat passes directly through the digestive system as an oil and is eliminated from the bowel in its oily undigested form.
Certain polysaccharides are non-absorbable and some polysaccharides have the side benefit of reducing lipid absorption by the body. Defatted rice germ polysaccharides and sulfated polysaccharides are also lipase inhibitors, which are high molecular weight compounds that do not appear to have any lactone moieties and seem to work by a different mechanism, binding the lipase and removing it from the digestive system when they are discharged from the digestive system. The super fiber Chitosan, which is a deacylated polysaccharide derived from shellfish chitan, has an ability to absorb fat and cholesterol, particularly in combination with vitamin C. Chitosan compositions may actually absorb up to 6 to 8 times their weight in fat and oils. While the polysaccharide from shellfish is similar to crude cellulose plant fiber, it has the ability to significantly bind fat in the digestive system as compared to plant fiber. Further, since polysaccharides, including those which do not preferentially bind oils over water, are not absorbed by the digestive systems of animals such as humans, non-human primates, dogs and cats, there is no caloric value to such polysaccharides and they pass through the such digestive systems unabsorbed and substantially intact. Examples of non-absorbable polysaccharides are polysaccharides having a molecular weight of greater than 8 kDa such as dextrans, molecular microcrystalline cellulose, wheat bran, oat bran, defatted rice germ, alginic acid, pectin, amylopectin, chitin, crude cellulose, argar, chitosan and the like.
There is a need in the art for non-absorbable lipase inhibitors, as well as for improved antiadiposity compositions and methods which do not require an absolute low-fat diet in order to lower the absorption of dietary fat as calories.
In one aspect the present invention relates to novel derivatives of lipase inhibitors which are non-absorbable compounds comprising at least one lipase inhibitor moiety and at least one non-absorbable polymeric moiety in the same molecule or salt. The lipase inhibitor moiety is preferably present in the non-absorbable compound in a weight ratio of from about 1:10 to about 1:60 with respect to the weight of the polymeric moiety, preferably from about 1:20 to about 1:40, and more preferably from about 1:25 to 1:35. In one aspect, such lipase inhibitors comprise at least one lipase inhibitor moiety (or moieties) linked directly or indirectly to such a polymeric moiety. The invention also includes pharmaceutical compositions comprising an effective amount of such lipase inhibitors in combination with a pharmaceutically acceptable carrier or diluent, which compositions may further comprise an effective amount of a lipophilic, non-absorbable biocompatible, pharmaceutically acceptable oil absorbing polymer.
In another aspect the present invention relates to novel salts of non-absorbable lipase inhibitors and a non-absorbable biocompatible, pharmaceutically acceptable oil absorbing polymer. The invention also includes pharmaceutical compositions comprising an effective amount of such lipase inhibitors in combination with a pharmaceutically acceptable carrier or diluent, which compositions may further comprise an effective amount of a lipophilic, non-absorbable biocompatible, pharmaceutically acceptable oil absorbing polymer.
In a preferred aspect the present invention relates to novel non-absorbable derivatives of a 1,3 oxetanone lipase inhibitor, which include at least one 1,3 oxetanone lipase inhibiting moiety that is covalently or non-covalently linked to a non-absorbable biocompatible, pharmaceutically acceptable polymer moiety to provide a novel lipase inhibitor compound. Preferred compounds have the dual function of inhibiting lipases and absorbing fat, in that the non-absorbable biocompatible, pharmaceutically acceptable polymer moiety of the novel lipase inhibitor will bind to fat, carry the bound fat with it through portions of the digestive system and cause the non-absorbed fat to be eliminated removed from the digestive system as undigested fat. The 1,3 oxetanone moiety that is derivatized directly or indirectly with the polymer moiety according to the invention may be initially an absorbable or non-absorbable moiety and is derivatized by directly or indirectly linking it to the polymer moiety to form a novel non-absorbable lipase inhibitor, preferably at the 5 hydroxyl position of a 1,3 oxetanone moiety.
In a preferred aspect the invention provides compounds having either non-covalent linkages of such two moieties or covalent linkages that are hydrolyzed or digested in the digestive system, providing that the lipase inhibiting 1,3 oxetanone derivative moiety that is released in the digestive system is substantially non-absorbable.
In another preferred aspect the invention provides compounds having either a non-covalent linkage of such two moieties or a covalent linkage that is not hydrolyzed or digested in the digestive system, whereby the lipase inhibiting 1,3 oxetanone derivative moiety remains linked to the polymer moiety via such non-covalent or covalent linkage and is not released in the digestive system.
In one aspect of the invention, an absorbable lipases such as esterastin moiety, tetrahydroestrastin, or a similar moiety, is rendered non-absorbable by coupling it directly or indirectly to a non-absorbable biocompatible, pharmaceutically acceptable polymer moiety, such as a polysaccharide, to render the lipase essentially non-absorbable by the digestive system of an animal such as a dog, cat, non-human primate or humans. Preferred absorbable lipase inhibitor moieties that are rendered non-absorbable by such coupling include at least one lipase inhibitor which is a member selected from the group consisting of esterastin, tetrahydro-esterastin (3,5-hydroxy-2-hexadeca-7,10-dienoic 1,3-lactone), 3,5-dihydroxy-2-hexylhexadeca-7,10-dienoic 1,3-lactone, 3,5-di-hydroxy-2-hexylhexadecanoic 1,3-lactone, and the like. Preferably, such lipase inhibitor is coupled to a non-absorbable biocompatible, pharmaceutically acceptable polymer moiety, such as a polysaccharide, to render the lipase non-absorbable by the digestive system of an animal such as a dog, cat, non-human primate or humans. Particularly preferred polysaccharides have at least one member selected from the group consisting of dextrans, molecular microcrystalline cellulose, wheat bran, oat bran, defatted rice germ, alginic acid, pectin, amylopectin, chitin, crude cellulose, argar, chitosan, a chitosan methylbenzoic acid ester ether derivative, and the like. Particularly preferred bound lipase inhibitors are lipase inhibitors bound via a derivatized group on the lipase such as a derivatized nitrogen, acid or alcohol group to a group on the polymer moiety such as a derivatized alcohol, acid or amino group. Preferably, a diether bridge is formed between the lipase inhibitor and the moiety, wherein the bridge is derived from an alcohol group on the lipase and an alcohol group on the moiety, each reacting with an etherizing bridging group. Also preferred are compounds wherein the oxetanone moiety is derivatized to provide an amino group which is further derivatized to form a carboxamide group, followed by linking the carboxamide group to an acid or alcohol group on the polymer moiety via a bridging group, which polymer moiety may have been derivativized in order to provide such an acid or alcohol group for attachment. Examples of such attachments are illustrated below by a preferred embodiment of the invention.
In another aspect the present invention relates to pharmaceutical compositions comprising a lipase inhibiting effective amount of at least one lipase inhibitor which is coupled to a digestively non-absorbable moiety. Preferred are such pharmaceutical compositions comprising an effective amount of a lipases coupled to a non-absorbable biocompatible, pharmaceutically acceptable polymer moiety, such as a polysaccharide, wherein the lipase is essentially non-absorbable by the digestive system of an animal such as a dog, cat, non-human primate or humans.
In still another aspect, the present invention relates to a method for treating adiposity or obesity by administering to a patient before a fat-contain meal, or up to one hour after such a meal is consumed, an amount of at least one lipase inhibitor which is bound to a non-absorbable polymer moiety in an amount effective to inhibit the absorption of up to one-third of the dietary fat in such a meal. In particular, a preferred method comprises administering at one lipase inhibitor which is a member selected from the group consisting of esterastin, tetrahydro-esterastin (3,5-hydroxy-2hexadeca-7,10-dienoic 1,3-lactone), 3,5-dihydroxy-2-hexylhexadeca-7,10-dienoic 1,3-lactone, 3,5-di-hydroxy-2-hexylhexadecanoic 1,3-lactone, and the like, wherein such lipase inhibitor is coupled to a non-absorbable biocompatible, pharmaceutically acceptable polymer moiety, such as a polysaccharide, to render the lipase non-absorbable by the digestive system of an animal such as a dog, cat, non-human primate or humans. Particularly preferred polysaccharides are at least one member selected from the group consisting of dextrans, molecular microcrystalline cellulose, wheat bran, oat bran, defatted rice germ, alginic acid, pectin, amylopectin, chitin, crude cellulose, argar, chitosan and the like. Particularly preferred bound lipase inhibitors are lipase inhibitors bound via a derivatized nitrogen, acid or alcohol group to a derivatized alcohol, acid or amino group on the polymer moiety.
A diether terminated bridge, or a terminal ether/terminal ester bridge, between the lipase inhibitor moiety and the polymer moiety which is derived from an alcohol group on the lipase inhibitor moiety and an alcohol group on the polymer moiety, respectively reacting with a bridging group is a preferred coupling of the lipase inhibitor to the moiety.
Another preferred bridge between the lipase inhibitor moiety and the polymer moiety includes at least one ether bridge formed from an alcohol group on the polymer moiety and at least one carboxamide bond. Further preferred are compound wherein at least one amino acid derivative is located in the bridge, and is bound directly or indirectly to the 5 hydroxyl position on the 1,3 oxetanone moiety via an ester linkage.
The preferred compounds also include their pharmaceutically acceptable isomers, hydrates, solvates, salts and prodrug derivatives.
In accordance with the present invention and as used herein, the following terms are defined with the following meanings, unless explicitly stated otherwise.
The term xe2x80x9calkenylxe2x80x9d refers to a trivalent straight chain or branched chain unsaturated aliphatic radical. The term xe2x80x9calkinylxe2x80x9d (or xe2x80x9calkynylxe2x80x9d) refers to a straight or branched chain aliphatic radical that includes at least two carbons joined by a triple bond. If no number of carbons is specified alkenyl and alkinyl each refer to radicals having from 2-12 carbon atoms.
The term xe2x80x9calkylxe2x80x9d refers to saturated aliphatic groups including straight-chain, branched-chain and cyclic groups having the number of carbon atoms specified, or if no number is specified, having up to 12 carbon atoms. The term xe2x80x9ccycloalkylxe2x80x9d as used herein refers to a mono-, bi-, or tricyclic aliphatic ring having 3 to 14 carbon atoms and preferably 3 to 7 carbon atoms.
The term xe2x80x9cbridging groupxe2x80x9d refers to a bifunctional chain or spacer group capable of reacting with one or more functional groups on a lipase inhibitor compound and then react with a second same or different functional group on a polymer compound in order to form a linked structure or conjugate between the two compounds. The bond formed between the bridging group and each of the two moieties is preferably of a type that is resistant to cleavage by the digestive environment when the linked lipase moiety would be absorbable upon cleavage of the bond. In one aspect, the bridging group is of the formula Xxe2x80x94Rxe2x80x94X, wherein R is a member selected from a straight-chained or branched alkyl group, a straight-chained or branched alkenyl group, a straight-chained or branched alkynyl group, a mono acyl group, a diacyl group and the like, which R portion of the chain may include a cycloalkyl or an aryl group, and X is a functionally reactive group such as a halogen, or acid group, under special reaction conditions as described hereinafter. Particularly preferred bridging groups form a diether, diacyl or ether/acyl bridge that is resistant to cleavage by the digestive environment. In one aspect bridging groups that are cleaved by the digestive group to release an essentially non-absorbable lipase inhibitor are preferred. Examples of alkylene dichloride bridging group forming compounds are dichloromethane, 1,2-dichloroethane, 1,2- and 1,3-dichloropropane, 1,2-, 1,3- and 1,4-dichlorobutane, and the like. Examples of acyldichloride bridging group forming compounds are oxalic acid dichloride, malonic acid dichloride, succinic acid dichloride, glutaric acid dichloride, adipic acid dichloride, pimelic acid dichloride, suberic acid dichloride, fumaric acid dichloride, malic acid dichloride, glutamic acid dichloride, terephthalic acid dichloride, isophthalic acid dichloride, haloalkylbenzoic acid, and the like. Other such bridging group reagents are compounds such as epichlorhydrin, phosphorus oxychloride, and diphosphoryl tetrachloride, and the like.
As used herein, the terms xe2x80x9ccarbocyclic ring structure xe2x80x9candxe2x80x9d C3-16 carbocyclic mono, bicyclic or tricyclic ring structurexe2x80x9d or the like are each intended to mean stable ring structures having only carbon atoms as ring atoms wherein the ring structure is a substituted or unsubstituted member selected from the group consisting of: a stable monocyclic ring which is aromatic ring (xe2x80x9carylxe2x80x9d) having six ring atoms; a stable monocyclic non-aromatic ring having from 3 to 7 ring atoms in the ring; a stable bicyclic ring structure having a total of from 7 to 12 ring atoms in the two rings wherein the bicyclic ring structure is selected from the group consisting of ring structures in which both of the rings are aromatic, ring structures in which one of the rings is aromatic and ring structures in which both of the rings are non-aromatic; and a stable tricyclic ring structure having a total of from 10 to 16 atoms in the three rings wherein the tricyclic ring structure is selected from the group consisting of: ring structures in which three of the rings are aromatic, ring structures in which two of the rings are aromatic and ring structures in which three of the rings are non-aromatic. In each case, the non-aromatic rings when present in the monocyclic, bicyclic or tricyclic ring structure may independently be saturated, partially saturated or fully saturated. Examples of such carbocyclic ring structures include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, adamantyl, cyclooctyl, [3.3.0]bicyclooctane, [4.3.0]bicyclononane, [4.4.0]bicyclodecane (decalin), 2.2.2]bicyclooctane, fluorenyl, phenyl, naphthyl, indanyl, adamantyl, or tetrahydronaphthyl (tetralin). Moreover, the ring structures described herein may be attached to one or more indicated pendant groups via any carbon atom which results in a stable structure. The termxe2x80x9csubstitutedxe2x80x9d as used in conjunction with carbocyclic ring structures means that hydrogen atoms attached to the ring carbon atoms of ring structures described herein may be substituted by one or more of the substituents indicated for that structure if such substitution(s) would result in a stable compound.
The term xe2x80x9carylxe2x80x9d which is included with the term xe2x80x9ccarbocyclic ring structurexe2x80x9d refers to an unsubstituted or substituted aromatic ring, substituted with one, two or three substituents selected from loweralkoxy, loweralkyl, loweralkylamino, hydroxy, halogen, cyano, hydroxyl, mercapto, nitro, thioalkoxy, carboxaldehyde, carboxyl, carboalkoxy and carboxamide, including but not limited to carbocyclic aryl, heterocyclic aryl, and biaryl groups and the like, all of which may be optionally substituted. Preferred aryl groups include phenyl, halophenyl, loweralkylphenyl, napthyl, biphenyl, phenanthrenyl and naphthacenyl.
The term xe2x80x9carylalkylxe2x80x9d which is included with the term xe2x80x9ccarbocyclic arylxe2x80x9d refers to one, two, or three aryl groups having the number of carbon atoms designated, appended to an alkyl group having the number of carbon atoms designated. Suitable arylalkyl groups include, but are not limited to, benzyl, picolyl, naphthylmethyl, phenethyl, benzyhydryl, trityl, and the like, all of which may be optionally substituted.
As used herein, the term xe2x80x9cheterocyclic ringxe2x80x9d or xe2x80x9cheterocyclic ring systemxe2x80x9d is intended to mean a substituted or unsubstituted member selected from the group consisting of stable monocyclic ring having from 5-7 members in the ring itself and having from 1 to 4 hetero ring atoms selected from the group consisting of N, O and S; a stable bicyclic ring structure having a total of from 7 to 12 atoms in the two rings wherein at least one of the two rings has from 1 to 4 hetero atoms selected from N, O and S, including bicyclic ring structures wherein any of the described stable monocyclic heterocyclic rings is fused to a hexane or benzene ring; and a stable tricyclic heterocyclic ring structure having a total of from 10 to 16 atoms in the three rings wherein at least one of the three rings has from 1 to 4 hetero atoms selected from the group consisting of N, O and S. Any nitrogen and sulfur atoms present in a heterocyclic ring of such a heterocyclic ring structure may be oxidized. Unless indicated otherwise the terms xe2x80x9cheterocyclic ringxe2x80x9d or xe2x80x9cheterocyclic ring systemxe2x80x9d include aromatic rings, as well as non-aromatic rings which can be saturated, partially saturated or fully saturated non-aromatic rings. Also, unless indicated otherwise the term xe2x80x9cheterocyclic ring systemxe2x80x9d includes ring structures wherein all of the rings contain at least one hetero atom as well as structures having less than all of the rings in the ring structure containing at least one hetero atom, for example bicyclic ring structures wherein one ring is a benzene ring and one of the rings has one or more hetero atoms are included within the term xe2x80x9cheterocyclic ring systemsxe2x80x9d as well as bicyclic ring structures wherein each of the two rings has at least one hetero atom. Moreover, the ring structures described herein may be attached to one or more indicated pendant groups via any hetero atom or carbon atom which results in a stable structure. Further, the term xe2x80x9csubstitutedxe2x80x9d means that one or more of the hydrogen atoms on the ring carbon atom(s) or nitrogen atom(s) of the each of the rings in the ring structures described herein may be replaced by one or more of the indicated substituents if such replacement(s) would result in a stable compound. Nitrogen atoms in a ring structure may be quaternized, but such compounds are specifically indicated or are included within the term xe2x80x9ca pharmaceutically acceptable saltxe2x80x9d for a particular compound. When the total number of O and S atoms in a single heterocyclic ring is greater than 1, it is preferred that such atoms not be adjacent to one another. Preferably, there are no more that 1 O or S ring atoms in the same ring of a given heterocyclic ring structure.
Examples of monocylic and bicyclic heterocylic ring systems, in alphabetical order, are acridinyl, azocinyl, benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzthiazolyl, benztriazolyl, benztetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazalinyl, carbazolyl, 4aH-carbazolyl, carbolinyl, chromanyl, chromenyl, cinnolinyl, decahydroquinolinyl, 2H,6H-1,5,2-dithiazinyl, dihydrofuro[2,3-b]tetrahydrofuran, furanyl, furazanyl, imidazolidinyl, imidazolinyl, imidazolyl, 1H-indazolyl, indolinyl, indolizinyl, indolyl, 3H-indolyl, isobenzofuranyl, isochromanyl, isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl (benzimidazolyl), isothiazolyl, isoxazolyl, morpholinyl, naphthyridinyl, octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, oxazolidinyl, oxazolyl, oxazolidinyl, pyrimidinyl, phenanthridinyl, phenanthrolinyl, phenazinyl, phenothiazinyl, phenoxathiinyl, phenoxazinyl, phthalazinyl, piperazinyl, piperidinyl, pteridinyl, purinyl, pyranyl, pyrazinyl, pyroazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pryidooxazole, pyridoimidazole, pyridothiazole, pyridinyl, pyridyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl, 2H-pyrrolyl, pyrrolyl, quinazolinyl, quinolinyl, 4H-quinolizinyl, quinoxalinyl, quinuclidinyl, tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl, 6H-1,2,5-thiadazinyl, 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thienothiazolyl, thienooxazolyl, thienoimidazolyl, thiophenyl, triazinyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,2,5-triazolyl, 1,3,4-triazolyl and xanthenyl. Preferred heterocyclic ring structures include, but are not limited to, pyridinyl, furanyl, thienyl, pyrrolyl, pyrazolyl, pyrrolidinyl, imidazolyl, indolyl, benzimidazolyl, 1H-indazolyl, oxazolinyl, or isatinoyl. Also included are fused ring and spiro compounds containing, for example, the above heterocylic ring structures.
As used herein the term xe2x80x9caromatic heterocyclic ring systemxe2x80x9d has essentially the same definition as for the monocyclic and bicyclic ring systems except that at least one ring of the ring system is an aromatic heterocyclic ring or the bicyclic ring has an aromatic or non-aromatic heterocyclic ring fused to an aromatic carbocyclic ring structure.
The terms xe2x80x9chaloxe2x80x9d or xe2x80x9chalogenxe2x80x9d as used herein refer to Cl, Br, F or I substituents. The term xe2x80x9chaloalkylxe2x80x9d, and the like, refer to an aliphatic carbon radicals having at least one hydrogen atom replaced by a Cl, Br, F or I atom, including mixtures of different halo atoms. Trihaloalkyl includes trifluoromethyl and the like as preferred radicals, for example.
The term xe2x80x9cmethylenexe2x80x9d refers to xe2x80x94CH2xe2x80x94.
The term xe2x80x9cpharmaceutically acceptable saltsxe2x80x9d includes salts of compounds derived from the combination of a compound and an organic or inorganic acid. These compounds are useful in both free base and salt form. In practice, the use of the salt form amounts to use of the base form; both acid and base addition salts are within the scope of the present invention.
xe2x80x9cPharmaceutically acceptable acid addition saltxe2x80x9d refers to salts retaining the biological effectiveness and properties of the free bases and which are not biologically or otherwise undesirable, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicyclic acid and the like.
xe2x80x9cPharmaceutically acceptable base addition saltsxe2x80x9d include those derived from inorganic bases such as sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Particularly preferred are the ammonium, potassium, sodium, calcium and magnesium salts. Salts derived from pharmaceutically acceptable organic nontoxic bases include salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-diethylaminoethanol, trimethamine, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purines, piperizine, piperidine, N-ethylpiperidine, polyamine resins and the like. Particularly preferred organic nontoxic bases are isopropylamine, diethylamine, ethanolamine, trimethamine, dicyclohexylamine, choline, and caffeine.
xe2x80x9cBiological propertyxe2x80x9d for the purposes herein means an in vivo effector or antigenic function or activity that is directly or indirectly performed by a compound of this invention that are often shown by in vitro assays. Effector functions include receptor or ligand binding, any enzyme activity or enzyme modulatory activity, any carrier binding activity, any hormonal activity, any activity in promoting or inhibiting adhesion of cells to an extracellular matrix or cell surface molecules, or any structural role. Antigenic functions include possession of an epitope or antigenic site that is capable of reacting with antibodies raised against it.
In the compounds of this invention, carbon atoms bonded to four non-identical substituents are asymmetric. Accordingly, the compounds may exist as diastereoisomers, enantiomers or mixtures thereof. The syntheses described herein may employ racemates, enantiomers or diastereomers as starting materials or intermediates. Diastereomeric products resulting from such syntheses may be separated by chromatographic or crystallization methods, or by other methods known in the art. Likewise, enantiomeric product mixtures may be separated using the same techniques or by other methods known in the art. Each of the asymmetric carbon atoms, when present in the compounds of this invention, may be in one of two configurations (R or S) and both are within the scope of the present invention.
In one aspect the present invention relates to novel derivatives of lipase inhibitors which are non-absorbable and have a lipase inhibitor moiety and polymeric moiety in the same molecule. The invention also includes pharmaceutical compositions comprising an effective amount of such lipase inhibitors in combination with a pharmaceutically acceptable carrier or diluent, and may further comprise an effective amount of a lipophilic, non-absorbable biocompatible, pharmaceutically acceptable oil absorbing polymer. The lipase inhibitor moiety is present in a weight ratio of from about 1:10 to about 1:60 with respect to the weight of the polymeric moiety, preferably from about 1:20 to about 1:40, and more preferably from about 1:25 to 1:35. In one aspect, such lipase inhibitors comprise at least one lipase inhibitor moiety (or moieties) linked directly or indirectly to such polymeric moiety.
In another aspect the present invention relates to novel salts of non-absorbable lipase inhibitors and a non-absorbable biocompatible, pharmaceutically acceptable oil absorbing polymer. The invention also includes pharmaceutical compositions comprising an effective amount of such lipase inhibitors in combination with a pharmaceutically acceptable carrier or diluent, which compositions may further comprise an effective amount of a lipophilic, non-absorbable biocompatible, pharmaceutically acceptable oil absorbing polymer.
In a preferred aspect the present invention relates to novel non-absorbable derivatives of a 1,3 oxetanone lipase inhibitor, which include at least one 1,3 oxetanone lipase inhibiting moiety that is covalently or non-covalently linked to a non-absorbable biocompatible, pharmaceutically acceptable polymer moiety to provide a novel lipase inhibitor compound. Preferred compounds have the dual function of inhibiting lipases and absorbing fat, in that the non-absorbable biocompatible, pharmaceutically acceptable polymer moiety of the novel lipase inhibitor will bind to fat, carry the bound fat with it through portions of the digestive system and cause the non-absorbed fat to be eliminated removed from the digestive system as undigested fat. The 1,3 oxetanone moiety that is derivatized directly or indirectly with the polymer moiety according to the invention may be initially an absorbable or non-absorbable moiety and is derivatized by directly or indirectly linking it to the polymer moiety to form a novel non-absorbable lipase inhibitor, preferably at the 5 hydroxyl position of a 1,3 oxetanone moiety.
In a preferred aspect the invention provides compounds having either non-covalent linkages of such two moieties or covalent linkages that are hydrolyzed or digested in the digestive system, providing that the lipase inhibiting 1,3 oxetanone derivative moiety that is released in the digestive system is substantially non-absorbable.
In another preferred aspect the invention provides compounds having either a non-covalent linkage of such two moieties or a covalent linkage that is not hydrolyzed or digested in the digestive system, whereby the lipase inhibiting 1,3 oxetanone derivative moiety remains linked to the polymer moiety via such non-covalent or covalent linkage and is not released in the digestive system.
In one aspect of the invention, an absorbable lipases such as esterastin moiety, tetrahydroestrastin, or a similar moiety, is rendered non-absorbable by coupling it directly or indirectly to a non-absorbable biocompatible, pharmaceutically acceptable polymer moiety, such as a polysaccharide, to render the lipase essentially non-absorbable by the digestive system of an animal such as a dog, cat, non-human primate or humans. Preferred absorbable lipase inhibitor moieties that are rendered non-absorbable by such coupling include at least one lipase inhibitor which is a member selected from the group consisting of esterastin, tetrahydro-esterastin (3,5-hydroxy-2-hexadeca-7,10-dienoic 1,3-lactone), 3,5-dihydroxy-2-hexylhexadeca-7,10-dienoic 1,3-lactone, 3,5-di-hydroxy-2-hexylhexadecanoic 1,3-lactone, and the like. Preferably, such lipase inhibitor is coupled to a non-absorbable biocompatible, pharmaceutically acceptable polymer moiety, such as a polysaccharide, to render the lipase non-absorbable by the digestive system of an animal such as a dog, cat, non-human primate or humans. Particularly preferred polysaccharides have at least one member selected from the group consisting of dextrans, molecular microcrystalline cellulose, wheat bran, oat bran, defatted rice germ, alginic acid, pectin, amylopectin, chitin, crude cellulose, argar, chitosan, a chitosan methylbenzoic acid ester ether derivative, and the like. Particularly preferred bound lipase inhibitors are lipase inhibitors bound via a derivatized group on the lipase such as a derivatized nitrogen, acid or alcohol group to a group on the polymer moiety such as a derivatized alcohol, acid or amino group. Preferably, a diether bridge is formed between the lipase inhibitor and the moiety, wherein the bridge is derived from an alcohol group on the lipase and an alcohol group on the moiety, each reacting with an etherizing bridging group. Also preferred are compounds wherein the oxetanone moiety is derivatized to provide an amino group which is further derivatized to form a carboxamide group, followed by linking the carboxamide group to an acid or alcohol group on the polymer moiety via a bridging group, which polymer moiety may have been derivativized in order to provide such an acid or alcohol group for attachment. Examples of such attachments are illustrated below by preferred embodiments of the invention.
A diether terminated bridge, or a terminal ether/terminal ester bridge, between the lipase inhibitor moiety and the polymer moiety which is derived from an alcohol group on the lipase inhibitor moiety and an alcohol group on the polymer moiety, respectively reacting with a bridging group is a preferred coupling of the lipase inhibitor to the moiety.
Another preferred bridge between the lipase inhibitor moiety and the polymer moiety includes at least one ether bridge formed from an alcohol group on the polymer moiety and at least one carboxamide bond. Further preferred are compound wherein at least one amino acid derivative is located in the bridge, and is bound directly or indirectly to the 5 hydroxyl position on the 1,3 oxetanone moiety via an ester linkage.
The preferred compounds also include their pharmaceutically acceptable isomers, hydrates, solvates, salts and prodrug derivatives.
A preferred aspect present invention relates to novel oxetanone derivatives of the formula I, as follows: 
wherein:
Xxe2x80x94Oxe2x80x94Q is an ether linkage wherein:
X of the ether linkage is a bridging group, and
Q of the ether linkage is a polysaccharide of a sufficient molecular weight or property that such polysaccharide is not absorbed by the digestive system of a mammal such as a dog, cat, non-human primate or a human primate, which polysaccharide is further defined below;
R is a member selected from the group consisting of:
a straight or branched chained C1-17-alkyl group which is saturated or optionally interrupted by up to eight double or triple bonds;
a straight or branched chained C1-17-alkyl group which is saturated or optionally interrupted by one or more members selected from the group consisting of an oxygen atom, a sulfur atom, a sulfonyl group or a sulfinyl group;
a straight or branched chained C1-17-alkyl group which is saturated or optionally interrupted by up to eight double or triple bonds and is interrupted in a position other than alpha to an unsaturated carbon atom by one or more members selected from the group consisting of an oxygen atom, a sulfur atom, a sulfonyl group or a sulfinyl group atoms;
phenyl substituted by 0-4 members selected from the group consisting of xe2x80x94C1-6-alkyloxy-C1-6-alkyl, xe2x80x94C1-6-alkylthio-C1-6-alkyl, xe2x80x94C1-6-alkyl-OH and xe2x80x94C1-6-alkyl-SH;
benzyl substituted by 0-4 members selected from the group consisting of xe2x80x94C1-6-alkyloxy-C1-6-alkyl, xe2x80x94C1-6-alkylthio-C1-6-alkyl, xe2x80x94C1-6-alkyl-OH and xe2x80x94C1-6-alkyl-SH;
biphenylene substituted by 0-6 members selected from the group consisting of xe2x80x94C1-6-alkyloxy-C1-6-alkyl, xe2x80x94C1-6-alkylthio-C1-6-alkyl, xe2x80x94C1-6-alkyl-OH and xe2x80x94C1-6-alkyl-SH;
phenoxyphenylene substituted by 0-6 members selected from the group consisting of xe2x80x94C1-6-alkyloxy-C1-6-alkyl, xe2x80x94C1-6-alkylthio-C1-6-alkyl, xe2x80x94C1-6-alkyl-OH and xe2x80x94C1-6-alkyl-SH;
phenylthiophenylene substituted by 0-6 members selected from the group consisting of xe2x80x94C1-6-alkyloxy-C1-6-alkyl, xe2x80x94C1-6-alkylthio-C1-6-alkyl, xe2x80x94C1-6-alkyl-OH and xe2x80x94C1-6-alkyl-SH;
phenyl-C1-6-alkyl-phenyl wherein 0-6 hydrogen atoms on one or more of the phenyl ring and xe2x80x94C1-6-alkyl- group is/are replaced independently by a member selected from the group consisting of xe2x80x94C1-6-alkyloxy-C1-6-alkyl, xe2x80x94C1-6-alkylthio-C1-6-alkyl, xe2x80x94C1-6-alkyl-OH and xe2x80x94C1-6-alkyl-SH;
R1 is a member selected from the group consisting of:
Hydrogen,
Ar,
Ar-C1-5-alkyl and
C1-10-alkyl interrupted by 0-3 members independently selected from the group consisting of an oxygen atom, a sulfur atom, a sulfinyl group, a sulfonyl group, a xe2x80x94N(xe2x80x94R4)xe2x80x94 group, a xe2x80x94C(xe2x95x90O)xe2x80x94N(xe2x80x94R4)xe2x80x94 group, and a xe2x80x94N(xe2x80x94R4)xe2x80x94C(xe2x95x90O)xe2x80x94 group, wherein 0xe2x80x943 carbon atoms of the C1-10-alkyl group can be substituted independently by a member selected from the group consisting of a hydroxy group, thiol group, C1-10-alkoxy group, a C1-10-alkylthio group, a xe2x80x94N(xe2x80x94R5, xe2x80x94R6) group, a xe2x80x94C(xe2x95x90O)xe2x80x94N(xe2x80x94R7, xe2x80x94R8) group, and a xe2x80x94N(xe2x80x94R9)xe2x80x94C(xe2x95x90O)xe2x80x94R10 group;
R2 is a member selected from the group consisting of:
hydrogen and C1-6-alkyl, or R2 taken with R1 forms a 4-6 membered saturated ring containing 0-4 nitrogen atoms wherein the ring may be substituted by 0-4 R11 groups;
R3 is a member selected from the group consisting of:
hydrogen, C1-6-alkyl and C1-6-alkanoyl,or R2 taken with R1 forms a 4-6 membered saturated ring;
R4-R10 are each independently a member selected from the group consisting of:
hydrogen and C1-6-alkyl;
n is an integer of 0-3;
and all pharmaceutically acceptable isomers, salts, hydrates, solvates and prodrug derivatives thereof.
A preferred compound according to formula I is a compound wherein X is a member selected from the group consisting of:
xe2x80x94(C(xe2x95x90O))0-1xe2x80x94Xaxe2x80x94,
wherein Xa is a member selected from the group consisting of:
a straight or branched chained divalent C1-17-alkylene group which is saturated or optionally interrupted by up to eight double or triple bonds;
a straight or branched chained divalent C1-17-alkylene group which is saturated or optionally interrupted by one or more members selected from the group consisting of:
an oxygen atom,
a sulfur atom,
a sulfonyl group,
a sulfinyl group,
a substituted or unsubstituted 6-10 member monocyclic or bicyclic aryl or heteroaryl group having from 1-4 ring hetero atoms selected from the group consisting of O, N, S,
a xe2x80x94NHxe2x80x94 group, wherein the hydrogen atom may be replaced with a C1-10alkyl group
a xe2x80x94C(xe2x95x90O)xe2x80x94 group,
a xe2x80x94NHxe2x80x94C(xe2x95x90O)xe2x80x94 group, wherein the hydrogen atom may be replaced with a C1-10 alkyl group and
a xe2x80x94C(xe2x95x90Oxe2x80x94NHxe2x80x94 group, wherein the hydrogen atom may be replaced with a C1-10 alkyl group
a straight or branched chained divalent C1-7-alkylene group which is saturated or optionally interrupted by up to eight double or triple bonds and is interrupted in a position other than alpha to an unsaturated carbon atom by one or more members selected from the group consisting optionally interrupted by one or more members selected from the group consisting of:
an oxygen atom,
a sulfur atom,
a sulfonyl group,
a sulfinyl group,
a substituted or unsubstituted 6-10 member monocyclic or bicyclic aryl or heteroaryl group having from 1-4 ring hetero atoms selected from the group consisting of O, N, S,
a xe2x80x94NHxe2x80x94 group, wherein the hydrogen atom may be replaced with a C1-10 alkyl group
a xe2x80x94C(xe2x95x90O)xe2x80x94 group,
a xe2x80x94NHxe2x80x94C(xe2x95x90O)xe2x80x94 group, wherein the hydrogen atom may be replaced with a C1-10alkyl group and
a xe2x80x94C(xe2x95x90O)xe2x80x94NHxe2x80x94 group, wherein the hydrogen atom may be replaced with a C1-10 alkyl group
divalent phenylene or divalent naphthylene substituted on the ring structure by 0-4 members selected from the group consisting of xe2x80x94C1-6-alkyloxy-C1-6-alkyl, xe2x80x94C1-6-alkylthio-C1-6-alkyl, xe2x80x94C1-6-alkyl-OH and xe2x80x94C1-6-alkyl-SH;
divalent biphenylene substituted by 0-6 members selected from the group consisting of xe2x80x94C1-6-alkyloxy-C1-6-alkyl, xe2x80x94C1-6-alkylthio-C1-6-alkyl, xe2x80x94C1-6-alkyl-OH and xe2x80x94C1-6-alkyl-SH;
phenoxyphenylene substituted by 0-6 members selected from the group consisting of xe2x80x94C1-6-alkyloxy-C1-6-alkyl, xe2x80x94C1-6-alkylthio-C1-6-alkyl, xe2x80x94C1-6-alkyl-OH and xe2x80x94C1-6-alkyl-SH;
divalent phenylthiophenylene substituted by 0-6 members selected from the group consisting of xe2x80x94C1-6-alkyloxy-C1-6-alkyl, xe2x80x94C1-6-alkylthio-C1-6-alkyl, xe2x80x94C1-6-alkyl-OH and xe2x80x94C1-6-alkyl-SH; and
and all pharmaceutically acceptable isomers, salts, hydrates, solvates and prodrug derivatives thereof.
A preferred groups of such compounds is compounds wherein R is xe2x80x94(CH2)3-6xe2x80x94CH3.
In certain aspects of this invention, compounds are provided which are useful as diagnostic reagents. In another aspect, the present invention includes pharmaceutical compositions comprising a pharmaceutically effective amount of the compounds of this invention and a pharmaceutically acceptable carrier. In yet another aspect, the present invention includes methods comprising using the above compounds and pharmaceutical compositions for preventing or treating disease states characterized by undesired lipid or fat absorption such as obesity, hyperlipaemia, atherosclerosis and ateioscherosis disorders of the blood coagulation process in mammals, or for stabalizing fats by preventing lipase function in stored fat products and samples. Optionally, the methods of this invention comprise administering the pharmaceutical composition in combination with an additional therapeutic agent such as an anticholesterol agent, appetite suppressant, metabolic stimulant and the like.
The preferred compounds also include their pharmaceutically acceptable isomers, hydrates, solvates, salts and prodrug derivatives.
The present invention also relates to a method for producing novel oxetanone derivatives of the formula: 
comprising reacting a compound which is a member selected from: 
wherein Y is a leaving group for an etherification reaction with another hydroxy group, with a compound of the formula: 
wherein Z is an OH or a halogen atom to form the ester linkage and yield a compound of the formula: 
or the intermediate 
(wherein the xe2x80x94OH group or the intermediate is reacted with an Xxe2x80x94Y producing group) to also yield a compound of the formula 
and further reacting the Xxe2x80x94Y group with an alcohol group of a polysaccharide to produce a compound with the Xxe2x80x94Oxe2x80x94Q group which has the following formula: 
as described above.
The present invention also relates to the process of forming a compound of the formula: 
wherein the X, Q, n and R, R1, R2 and R3 groups are defined as above comprising reacting a Xxe2x80x94Y producing group with a free hydroxy group of a linear carbon chain atom of a lipase inhibitor compound derived from a microbial source, wherein the lipase inhibitor compound is a compound of the formula: 
and the unsubstituted free beta-hydroxy group that reacts with the Xxe2x80x94Y forming group is obtained by hydrolyzing an ester side chain of the lipase inhibitor or metabolite of a microbe to produce a free xe2x80x94OH group on, wherein naturally occuring unsaturated bonds of the R and R1 groups of the naturally occurring lipase inhibitor may optionally be reduced before the ester side chain is hydrolyzed to form the xe2x80x94OH group, prior to reaction of the xe2x80x94OH group with the Xxe2x80x94Y forming group or after the xe2x80x94OH group is reacted with the Xxe2x80x94Y forming group to produce a compound of the formula: 
and further reacting the Xxe2x80x94Y group with an alcohol group of a polysaccharide to produce a compound with the Xxe2x80x94Oxe2x80x94Q group which has the following formula: 
as described above.
In one embodiment the present invention provides a pharmaceutical composition comprising at least one pharmaceutically acceptable carrier exipient and an amount of at least one of the above described compounds according to the invention in a therapeutically effective amount with respect to limiting or preventing the absorption of some dietary fat. In a preferred embodiment, the pharmaceutical composition comprises a therapeutically effective amount of slow-release lipoprotein lipase, preferably from a microbial or plant source, which selectively hydrolyzes terminal triglyceride groups in combination with an oil absorbing effective amount of polysaccharide such as chitosan, wherein the lipoprotein lipase is present in a ratio of less that 25% with respect to the oil absorbing polysaccharide.
In another embodiment the present invention provides a pharmaceutical composition comprising at least one pharmaceutically acceptable carrier exipient, an amount of at least one of the above described compounds according to the invention in a therapeuticallly effective amount with respect to limiting or preventing the absorption of some dietary fat, and an oil absorbing effective amount of polysaccharide such as chitosan, wherein such lipase inhibitor is selectively effective to inhibit lipases other than lipases involved in the hydrolysis of terminal triglyceride groups and such lipase inhibitor does not substantially inhibit the absorption of vitamins A, D and E.
In another embodiment the present invention provides a method of using such compounds and pharmaceutical compositions as therapeutic agents for disease states in a mammal having at least one disorder that is due to undesired absorption of dietary fat or for reducing the effective caloric intake of a mammal who consumes dietary fat, which method may be useful in the treatment of undesired weight gain or obesity.
Particularly preferred Q groups for the above compounds are non-absorbable biocompatable, pharmaceutically acceptable polymers, such as a polysaccharide, which are preferably lipophilic and bind to fat and are non-absorbable by the digestive system of an animal such as a dog, cat, non-human primate or humans. Particularly preferred polysaccharides have at least one member selected from the group consisting of dextrans, molecular microcrystalline cellulose, wheat bran, oat bran, defatted rice germ, alginic acid, pectin, amylopectin, chitin, crude cellulose, argar, chitosan and the like. Particularly preferred Q groups have an alcohol, acyl group or amino group, or can be derivitized to present such an alcohol, acyl or amino group, which can be bound to the R moiety or the Xxe2x80x94Rxe2x80x94X group Preferably, at least one and or two ether bridges can be formed between a lipase inhibitor moiety and the polymer moiety via an Xxe2x80x94Rxe2x80x94X bridge, wherein the bridge is derived from an alcohol group or amine group on the lipase and an alcohol group, acyl group or amino group on the Q group.
The lipase inhibitor compounds, polymer moieties and bridging groups of the present invention may be synthesized or readily obtained from commercially available sources. Polymer bridging groups, bridge coupling processes and compound purification methods are described and referenced in standard textbooks, particularly the coupling of alcohol groups via diether bridges, ether/ester bridges, ether/ketone bridges and the like. Standard polymer textbooks reference typical bifunctional bridging groups and coupling procedures.
Starting materials used in any of these methods are commercially available from chemical vendors such as Aldrich, Sigma, Nova Biochemicals, Bachem Biosciences, and the like, or may be readily synthesized by known procedures.
Reactions are carried out in standard laboratory glassware and reaction vessels under reaction conditions of standard temperature and pressure, except where otherwise indicated.
During the synthesis of these compounds, the functional groups may be protected by blocking groups to prevent cross reaction during the coupling procedure. Examples of suitable blocking groups and their use are described in xe2x80x9cThe Peptides: Analysis, Synthesis, Biologyxe2x80x9d, Academic Press, Vol. 3 (Gross, et al., Eds., 1981) and Vol. 9 (1987), the disclosures of which are incorporated herein by reference.
Lipase inhibitor moieties having a free hydroxy group such as tetrahydro-esterastin (3,5-hydroxy-2-hexadeca-7,10-dienoic 1,3-lactone), 3,5-dihydroxy-2-hexylhexadeca-7,10-dienoic 1,3-lactone, 3,5-di-hydroxy-2-hexylhexadecanoic 1,3-lactone, and the like, are easily coupled to a polymer moiety having free hydroxy groups such as cellulose, chitosan and other polysaccharides having free hydroxyl groups. One or both of the lipase inhibitor moiety and the polymer moiety may be derivitized to form part of the linking bridge prior to reacting with the other moiety. For example, the lipase inhibitor molecule may be condensed with a dihalide group or a terminal halide terminal acyl (acyl group may be protected with an acid group) to form an ether or ester linkage and then condensed with a polymer moiety having a free hydroxyl group as shown in polysaccharide chemistry. In one procedure a polymer moiety such as chitosan can be reacted with a compound such as a halomethylbenzoic acid ester, or the like, and de-esterified to present a free acid group which may be reacted to form a ketone, carboxamide or the like with the derivitized lipase inhibitor moiety. In one preferred aspect of the invention, one of the two moieties is reacted with an asymmetrical dihalide bridging group, such as an alkylene dihalide (e.g., 1,2-bromochloroethyl, 1,3-bromochloropropyl, and the like) in a molar ratio of 1:1 to etherize a free hydroxyl groups, replace a hydrogen atom on an amino group, or from a ketone with an acid group, and the resulting intermediate can then be reacted with the polymer moiety to form a ether group with a free alcohol group, replace a nitrogen atom on a amino group or form a ketone with an acid group. Particularly preferred polymer moieties are polysaccharides having multiple free hydroxyl group which after coupling may optionally be sulfonated to render the lipase moiety itself a lipase inhibitor compound. Etherification, amination and ketone formation procedures are well-known in the art and well within the routine skill of the ordinary practitioner. Further, other bridging groups and the techniques for binding a compound having a reactive functional group to a polymer moiety are well-known in the art. The preferred compounds also include their pharmaceutically acceptable isomers, hydrates, solvates, salts and prodrug derivatives.
The bridging group refers to a bifunctional chain or spacer group capable of reacting with one or more functional groups on a lipase inhibitor compound and then react with a second same or different functional group on a polymer compound in order to form a linked structure or conjugate between the two compounds. The bond formed between the bridging group and each of the two compounds is preferably of a type that is resistant to cleavage by the digestive environment. In one aspect, the bridging group is of the formula Xxe2x80x94Rxe2x80x94X, wherein R is a member selected from a straight-chained or branched alkyl group, a straight-chained or branched alkenyl group, a straight-chained or branched alkynyl group, a mono acyl group, a diacyl group and the like, and X is a functionally reactive group such as a halogen, under special reaction conditions as described hereinafter. Particularly preferred bridging groups form a diether or diacyl bridge that is resistant to cleavage by the digestive environment.
Examples of alkylene dichloride bridging group forming compounds are dichloromethane, 1,2-dichloroethane, 1,2- and 1,3-dichloropropane, 1,2-, 1,3- and 1,4-dichlorobutane, 1,2-bromochloroethane, 1,2-and 1,3-bromochloropropane, and the like.
Examples of acyldichloride bridging group forming compounds are oxalic acid dichloride, malonic acid dichloride, succinic acid dichloride, glutaric acid dichloride, adipic acid dichloride, pimelic acid dichloride, suberic acid dichloride, fumaric acid dichloride, malic acid dichloride, glutamic acid dichloride, terephthalic acid dichloride, isophthalic acid dichloride, and the like.
Examples of haloacyl bridging groups include chloromethylbenzoic acid or an ester thereof, 3-bromopropanoic acid, 2-chloroacetic acid, and the like.
Other such bridging group reagents are compounds such as epichlorhydrin, phosphorus oxychloride, and diphosphoryl tetrachloride, and the like.
Preferred bridging groups are dihalide groups terminated with a bromo and a chlorine group, or groups terminated with an acyl group and a halogen group. The reaction is performed by the slow addition of a bifunctional reagent such as a diacyl dichloride, an alkylene dichloride, or a bromosubstituted acyl ester compound, dissolved in a substantially water immiscible organic solvent, to an alkaline aqueous solution of the lipase inhibitor in a substantially 1:1 molecular ratio. The reaction proceeds at the interface between the two immiscible solutions to provide an interfacial condensation and produce the sucrose derivative or analogue. It has been discovered that this reaction at the interface of the organic solution and the aqueous solution imparts a specificity to the reaction for primary alcohol groups of the polysaccharide. It should be understood that equivalent reactants such as diepoxides and halohydrocarbyloxiranes such as epichlorohydrin also react in the process to provide new and useful ether bridges.
By appropriate selection of the type of bridging group reactant, different structural groups with various chemical properties can be incorporated into the resulting bridge and various types of lipase inhibitors can be connected to a nonabsorbable polymer moiety, such as a polysaccharide, and preferably to chitosan. Reaction temperatures and other reactions conditions, as well are reactant proportions are well within the skill of the ordinary polymer chemist practitioner. Other groups and modifications will be apparent to one of ordinary skill in the art from the above discussion.
The lipase inhibitor functionality of the coupled lipase inhibitors may be determined by well-known lipase inhibitor assays. A therapeutically effective amount of the bound lipase inhibitor may be administered to a patient. Additional fat binding polymers may optionally be added to the composition.
In one aspect, the present invention provides a sports drink, snack, nutrient supplement, food or power which may be formulated to contain a lipase inhibiting therapeutically effective amount of the lipase inhibitor composition according to the invention.
In another aspect the present invention relates to pharmaceutical compositions comprising a lipase inhibiting effective amount of at least one lipase inhibitor which is coupled to a digestively non-absorbable moiety. Preferred are such pharmaceutical compositions comprising an effective amount of a lipases coupled to a non-absorbable biocompatable, pharmaceutically acceptable polymer moiety, such as a polysaccharide, wherein the lipase is essentially non-absorbable by the digestive system of an animal such as a dog, cat, non-human primate or humans. The pharmaceutical composition can be administrated to a patent prior to or within one hour of consuming a fat-containing meal to prevent absorption of up to one-third of the dietary fat consumed at the meal.
In still another aspect, the present invention relates to a method for treating adiposity or obesity by administering to a patient before a fat-contain meal, or up to one hour after such a meal is consumed, an amount of at least one lipase inhibitor which is bound to a non-absorbable polymer moiety in an amount effective to inhibit the absorption of up to one-third of the dietary fat in such a meal. In particular, a preferred method comprises administering at one lipase inhibitor which is a member selected from the group consisting of esterastin, tetrahydro-esterastin (3,5-hydroxy-2hexadeca-7,10-dienoic 1,3-lactone), 3,5-dihydroxy-2-hexylhexadeca-7,10-dienoic 1,3-lactone, 3,5-di-hydroxy-2-hexylhexadecanoic 1,3-lactone, and the like, wherein such lipase inhibitor is coupled to a non-absorbable biocompatable, pharmaceutically acceptable polymer moiety, such as a polysaccharide, to render the lipase non-absorbable by the digestive system of an animal such as a dog, cat, non-human primate or humans. Particularly preferred polysaccharides are at least one member selected from the group consisting of dextrans, molecular microcrystalline cellulose, wheat bran, oat bran, defatted rice germ, alginic acid, pectin, amylopectin, chitin, crude cellulose, argar, chitosan and the like. Particularly preferred bound lipase inhibitors are lipase inhibitors bound via a derivatized nitrogen, acid or alcohol group to a derivatized alcohol, acid or amino group on the polymer moiety. A diether bridge between the lipase inhibitor and the moiety which is derived from an alcohol group on the lipase and an alcohol group on the moiety, respectively reacting with a bridging group is the preferred coupling of the lipase inhibitor to the moiety.
The compounds of this invention may be isolated as the free acid or base or converted to salts of various inorganic and organic acids and bases. Such salts are within the scope of this invention. Non-toxic and physiologically compatible salts are particularly useful although other less desirable salts may have use in the processes of isolation and purification.
Numerous methods are useful for the preparation of the salts described above and are known to those skilled in the art. For example, free acid or free base forms of a compound of one of the above compounds can be reacted with one or more molar equivalents of the desired acid or base in a solvent or solvent mixture in which the salt is insoluble, or in a solvent like water after which the solvent is removed by evaporation, distillation or freeze drying.
Alternatively, the free acid or base form of the product may be passed over an ion exchange resin to form the desired salt or one salt form of the product may be converted to another using the same general process.
This invention also encompasses prodrug derivatives of the compounds contained herein. The term xe2x80x9cprodrugxe2x80x9d refers to a pharmacologically inactive derivative of a parent drug molecule that requires biotransformation, either spontaneous, acid/base reaction, or enzymatic, within the organism to release the active drug. Prodrugs are variations or derivatives of the compounds of this invention which have groups cleavable under digestive system conditions. Prodrugs become the compounds of the invention which are pharmaceutically active in vivo, when they undergo solvolysis under physiological conditions or undergo enzymatic degradation. Prodrug compounds of this invention may be called single, double, triple etc., depending on the number of biotransformation steps required to release the active drug within the organism, and indicating the number of functionalities present in a precursor-type form. Prodrug forms often offer advantages of solubility, digestive compatibility, or delayed release in the mammalian organism (see, Bundgard, Design of Prodrugs, pp. 7-9, 21-24, Elsevier, Amsterdam 1985 and Silverman, The Organic Chemistry of Drug Design and Drug Action, pp. 352-401, Academic Press, San Diego, Calif., 1992). Prodrugs commonly known in the art include acid derivatives well known to practitioners of the art, such as, for example, esters prepared by reaction of the parent acids with a suitable alcohol, or amides prepared by reaction of the parent acid compound with an amine, or basic groups reacted to form an acylated base derivative. Moreover, the prodrug derivatives of this invention may be combined with other features herein taught to enhance bioavailability.
Formulations of the compounds of this invention are prepared for storage or administration by mixing the compound having a desired degree of purity with physiologically acceptable carriers, excipients, stabilizers etc., and may be provided in sustained release or timed release formulations. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical field, and are described, for example, in Remington""s Pharmaceutical Sciences, Mack Publishing Co., (A. R. Gennaro edit. 1985). Such materials are nontoxic to the recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, acetate and other organic acid salts, antioxidants such as ascorbic acid, low molecular weight (less than about ten residues) peptides such as polyarginine, proteins, such as serum albumin, gelatin, or immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidinone, amino acids such as glycine, glutamic acid, aspartic acid, or arginine, monosaccharides, disaccharides, and other carbohydrates including cellulose or its derivatives, glucose, mannose or dextrins, chelating agents such as EDTA, sugar alcohols such as mannitol or sorbitol, counterions such as sodium and/or nonionic surfactants such as Tween, Pluronics or polyethyleneglycol.
Dosage formulations of the compounds of this invention to be used for therapeutic administration must be sterile. Sterility is readily accomplished by filtration through sterile membranes such as 0.2 micron membranes, or by other conventional methods. Formulations typically will be stored in lyophilized form or as an aqueous solution. The pH of the preparations of this invention typically will be 3-11, more preferably 5-9 and most preferably 7-8. It will be understood that use of certain of the foregoing excipients, carriers, or stabilizers will result in the formation of cyclic polypeptide salts.
Therapeutically effective dosages may be determined by either in vitro or in vivo methods. For each particular compound of the present invention, individual determinations may be made to determine the optimal dosage required. The range of therapeutically effective dosages will be influenced by the route of administration, the therapeutic objectives and the condition of the patient. Accordingly, it may be necessary for the therapist to titer the dosage and modify the means of administration as required to obtain the optimal therapeutic effect. The determination of effective dosage levels, that is, the dosage levels necessary to achieve the desired result, will be readily determined by one skilled in the art. Typically, applications of compound are commenced at lower dosage levels, with dosage levels being increased until the desired effect is achieved.
The compounds of the invention can be administered orally in an effective amount within the dosage range of about 10 to 400 mg/kg, preferably about 50 to 300 mg/kg and more preferably about 100 to 200 mg/kg per fat containing meal on a regimen in a single or 2 to 4 divided daily doses. A preferred dosage is an amount (e.g. about 100 to 200 mg/kg) that has a similar lipase inhibiting effect to the lipase inhibition of 120 mg (approximately 1-2 mg/kg dosage) of orally taken Orlistat. The determination of such equivalent lipase inhibition can be determined via well-known lipase inhibition assays, and may be either an in vivo assay, an in vitro assay, or both. The fat absorption properties of the lipophilic lipase inhibitor of the invention can be observed by comparing the amount of anal oil discharged in a patient taking an lipase inhibitor equivalent amount of the lipase inhibitor according to the invention as compared to a patient taking only Orlistat. The grooming of mice with anal oil is one comparison as compared to Orlistat or the actual comparison of anal discharge in animals or patients also will show a reduction in the amount of oily anal discharge when a lipophilic lipase inhibitor according to the invention is administered.
Typically, about 500 mg to 2 g of a compound or mixture of compounds of this invention, as the free acid or base form or as a pharmaceutically acceptable salt, is compounded with a physiologically acceptable vehicle, carrier, excipient, binder, preservative, stabilizer, dye, flavor etc., as called for by accepted pharmaceutical practice. The amount of active ingredient in these compositions is such that a suitable dosage in the range indicated is obtained.
Typical adjuvants which may be incorporated into tablets, capsules and the like are binders such as acacia, corn starch or gelatin, and excipients such as microcrystalline cellulose, disintegrating agents like corn starch or alginic acid, lubricants such as magnesium stearate, sweetening agents such as sucrose or lactose, or flavoring agents. When a dosage form is a capsule, in addition to the above materials it may also contain liquid carriers such as water, saline, or a fatty oil. Other materials of various types may be used as coatings or as modifiers of the physical form of the dosage unit. Sterile compositions for injection can be formulated according to conventional pharmaceutical practice. For example, dissolution or suspension of the active compound in a vehicle such as an oil or a synthetic fatty vehicle like ethyl oleate, or into a liposome may be desired. Buffers, preservatives, antioxidants and the like can be incorporated according to accepted pharmaceutical practice.
In certain aspects of this invention, compounds are provided which are useful as diagnostic reagents to determine lipase activity. In another aspect, the present invention includes pharmaceutical compositions comprising a pharmaceutically effective amount of the compounds of this invention and a pharmaceutically acceptable carrier. In yet another aspect, the present invention includes methods comprising using the above compounds and pharmaceutical compositions for preventing or treating disease states characterized by undesired lipid or fat absorption such as obesity, hyperlipaemia, atherosclerosis and ateioscherosis disorders of the blood coagulation process in mammals, or for stabilizing fats by preventing lipase function in stored fat products and samples. Optionally, the methods of this invention comprise administering the pharmaceutical composition in combination with an additional therapeutic agent such as an anticholesterol agent, appetite suppressant, metabolic stimulant and the like.
The preferred compounds also include their pharmaceutically acceptable isomers, hydrates, solvates, salts and prodrug derivatives.
In one embodiment the present invention provides a pharmaceutical composition comprising at least one pharmaceutically acceptable carrier excipient and an amount of at least one of the above described compounds according to the invention in a therapeutically effective amount with respect to limiting or preventing the absorption of some dietary fat. In a preferred embodiment, the pharmaceutical composition comprises a therapeutically effective amount of slow-release lipoprotein lipase, preferably from a microbial or plant source, which selectively hydrolyzes terminal triglyceride groups in combination with an oil absorbing effective amount of polysaccharide such as chitosan, wherein the lipoprotein lipase is present in a ratio of less that 25% with respect to the oil absorbing polysaccharide.
In another embodiment the present invention provides a pharmaceutical composition comprising at least one pharmaceutically acceptable carrier excipient, an amount of at least one of the above described compounds according to the invention in a therapeutically effective amount with respect to limiting or preventing the absorption of some dietary fat, and an oil absorbing effective amount of polysaccharide such as chitosan, wherein such lipase inhibitor is selectively effective to inhibit lipases other than lipases involved in the hydrolysis of terminal triglyceride groups and such lipase inhibitor does not substantially inhibit the absorption of vitamins A, D and E.
In another embodiment the present invention provides a method of using such compounds and pharmaceutical compositions as therapeutic agents for disease states in a mammal having at least one disorder that is due to undesired absorption of dietary fat or for reducing the effective caloric intake of a mammal who consumes dietary fat, which method may be useful in the treatment of undesired weight gain or obesity.
The compounds of this invention also find utility as intermediates for producing therapeutic agents or as therapeutic agents for disease states in mammals which have disorders that are due to undesired absorption of dietary fat. Methods for making starting materials may be found in U.S. Pat. No. 4,931,463, which is incorporated fully herein. Preferred oxetanones of the invention are compounds wherein R is methyl, ethyl, propyl, hexyl, decyl, hexadecyl, allyl and benzyl, and most preferably hexyl; R1 is hydrogen, methyl, ethyl, propyl, 2-butyl, isobutyl, benzyl and methylthio-ethyl, most preferably, hydrogen or isobutyl; R2 is hydrogen, methyl or ethyl, most preferably hydrogen; n is 0 or 1, and when t is 1, then X is preferably attached to N via an amino acid, such as valine, alanine and the like, preferably alanine; and R3 is preferably a straight or branched chained C1-17-alkyl group which is saturated or optionally interrupted by up to eight double or triple bonds, or R3 is a straight or branched chained C1-17-alkyl group which is saturated or optionally interrupted by one or more members selected from the group consisting of an oxygen atom, a sulfur atom, a sulfonyl group or a sulfinyl group or R3 is a straight or branched chained C1-17-alkyl group which is saturated or optionally interrupted by up to eight double or triple bonds and is interrupted in a position other than alpha to an unsaturated carbon atom by one or more members selected from the group consisting of an oxygen atom, a sulfur atom, a sulfonyl group or a sulfinyl group atoms.
The compounds produced according to the present invention may also be used as intermediates in the formation of compounds that may be administered in combination or in concert with other therapeutic or diagnostic agents. In certain preferred embodiments, the compounds produced by the intermediates according to the present invention may be co-administered along with other compounds typically prescribed for these conditions according to generally accepted medical practice such as other dietary maintenance medicaments and for diseases related to or impacted by the absorption of dietary fat. The compounds produced from the intermediates according to the present invention may act in a synergistic fashion with other such medicaments. Such compounds may also allow for reduced doses of other cholesterol inhibiting, appetite inhibiting and metabolic stimulating medicaments, and the like. Such compounds can be utilized in vivo, ordinarily in mammals such as primates, (non-human and humans), sheep, horses, cattle, pigs, dogs, cats, rats and mice, or in vitro.
The starting materials used in above processes are commercially available from chemical vendors such as Aldrich, Sigma, Lancaster, TCI, and the like, or may be readily synthesized by known procedures, for example, by using procedures such as indicated above.
Reactions are carried out in standard laboratory glassware and reaction vessels under reaction conditions of standard temperature and pressure, except where otherwise indicated, or is well-known in literature available in the art. Further, the above procedures of the claimed invention processes my be carried out on a commercial scale by utilizing reactors and standard scale-up equipment available in the art for producing large amounts of compounds in the commercial environment. Such equipment and scale-up procedures are well-known to the ordinary practitioner in the field of commercial chemical production.
During the synthesis of these compounds, amino or acid functional groups may be protected by blocking groups to prevent undesired reactions with the amino group or acid group during certain procedures. Procedures for such protection and removal of protecting groups are routine in this art and well-known to the ordinary practitioner in this field.
Three non-limiting exemplary synthesis schemes were shown above, which are each a preferred embodiment of the invention, comprise the process steps outlined above which may also include further initial starting steps such as those set forth in J. Med. Chem., Vol. 15, No. 8 (1972) or further processing steps which modify the amino group to comprise a desired functional group such as groups described in the lipase inhibiting field. Amino coupling reactions are well-known in the art. Moreover, specific steps that are set forth in the preferred embodiment reaction scheme described above. The reaction products are isolated and purified by conventional methods, typically by solvent extraction into a compatible solvent. Preferred solvents are lower alkane ethers and alcohols; ethyl ether and isopropyl alcohol are preferred for solvent extraction or recrystallization procedures. Esters of carboxylic acid side groups may be formed that permit selective separation of the R and S enantiomers by solvent extraction or recrystallization. D-alaninol is the preferred enantiomer resolving agent, but other resolving agents or analogous procedures may be used, e.g., tartartic acid derivatives and the like. The products may be further purified by column chromatography or other appropriate methods.
As is clear from the above formulae and the discussion above, by using the above reactions racemic chromane acetic acid is obtained which may optionally be resolved to produce a racemic mixture enriched in either the R or S enantiomers or completely resolved into a substantially pure composition of one of the enantiomers. The literature in this field describes examples of conventional processes whereby the enantiomers may be resolved.
The above compounds produced according to the above invention may be isolated and further reacted to substitute a desired group for one or more of the hydrogen atoms on an amino group, on a free hydroxyl group or on a free acyl group by a coupling reaction with the desired group.
The compounds of this invention may be isolated as the free acid or base or converted to salts of various inorganic and organic acids and bases. Such salts are within the scope of this invention. Non-toxic and physiologically compatible salts are particularly useful although other less desirable salts may have use in the processes of isolation and purification.
A number of methods are useful for the preparation of the salts described above and are known to those skilled in the art. For example, reaction of the free acid or free base form of a compound of the structures recited above with one or more molar equivalents of the desired acid or base in a solvent or solvent mixture in which the salt is insoluble, or in a solvent like water after which the solvent is removed by evaporation, distillation or freeze drying. Alternatively, the free acid or base form of the product may be passed over an ion exchange resin to form the desired salt or one salt form of the product may be converted to another using the same general process.
Diagnostic applications of the compounds of this invention will typically utilize formulations such as solution or suspension. In the management of undesired fat absorption the compounds of this invention may be utilized in compositions such as tablets, capsules or elixirs for oral administration, sterile solutions or suspensions, and the like, or incorporated into shaped articles. Subjects in need of treatment (typically mammalian) using the compounds of this invention can be administered dosages that will provide optimal efficacy. The dose and method of administration will vary from subject to subject and be dependent upon such factors as the type of mammal being treated, its sex, weight, diet, concurrent medication, overall clinical condition, the particular compounds employed, the specific use for which these compounds are employed, and other factors which those skilled in the medical arts will recognize.
Formulations of the compounds of this invention are prepared for storage or administration by mixing the compound having a desired degree of purity with physiologically acceptable carriers, excipients, stabilizers etc., and may be provided in sustained release or timed release formulations. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical field, and are described, for example, in Remington""s Pharmaceutical Sciences, Mack Publishing Co., (A. R. Gennaro edit. 1985). Such materials are nontoxic to the recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, acetate and other organic acid salts, antioxidants such as ascorbic acid, low molecular weight (less than about ten residues) peptides such as polyarginine, proteins, such as serum albumin, gelatin, or immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidinone, amino acids such as glycine, glutamic acid, aspartic acid, or arginine, monosaccharides, disaccharides, and other carbohydrates including cellulose or its derivatives, glucose, mannose or dextrins, chelating agents such as EDTA, sugar alcohols such as mannitol or sorbitol, counterions such as sodium and/or nonionic surfactants such as Tween, Pluronics or polyethyleneglycol.
Dosage formulations of the compounds of this invention to be used for therapeutic administration must be sterile. Sterility is readily accomplished by filtration through sterile membranes such as 0.2 micron membranes, or by other conventional methods. Formulations typically will be stored in lyophilized form or as an aqueous solution. The pH of the preparations of this invention typically will be between 3 and 11, more preferably from 5 to 9 and most preferably from 7 to 8. It will be understood that use of certain of the foregoing excipients, carriers, or stabilizers will result in the formation of cyclic polypeptide salts. While the preferred route of administration is by oral tablets, capsules or other unit dose mechanisms, such as liquids, other methods of administration are also anticipated such as in food stuffs, employing a variety of dosage forms. The compounds of this invention are desirably incorporated into food articles which may include fats to prevent their absorption.
The compounds of this invention may also be coupled with suitable polymers to enhance their therapeutic effects. Such polymers can include lipophilic polymers, such as polysaccharides and the like.
Therapeutically effective dosages may be determined by either in vitro or in vivo methods. For each particular compound of the present invention, individual determinations may be made to determine the optimal dosage required. The range of therapeutically effective dosages will naturally be influenced by the route of administration, the therapeutic objectives, and the condition of the patient. For routes of administration, the lipase inhibitor activity, in view of the amount of fat consumed, must be individually determined for each inhibitor by methods well known in pharmacology. Accordingly, it may be necessary for the therapist to titer the dosage and modify the route of administration as required to obtain the optimal therapeutic effect. The determination of effective dosage levels, that is, the dosage levels necessary to achieve the desired result, will be within the ambit of one skilled in the art. Typically, applications of compound are commenced at lower dosage levels, with dosage levels being increased until the desired effect is achieved.
Typically, about 500 mg to 3 g of a lipase inhibitor compound or mixture of lipase inhibitor compounds of this invention, as the free acid or base form or as a pharmaceutically acceptable salt, is compounded with a physiologically acceptable vehicle, carrier, excipient, binder, preservative, stabilizer, dye, flavor etc., as called for by accepted pharmaceutical practice. The amount of active ingredient in these compositions is such that a suitable dosage in the range indicated is obtained. The addition, one or more other therapeutic ingredients such as a fat absorbing polysaccharide or fiber, a fat-specific lipase inhibitor or lipase, as well as other dietary agents may be utilized in therapeutically effective amounts.
Typical adjuvants which may be incorporated into tablets, capsules and the like are a binder such as acacia, corn starch or gelatin, and excipient such as microcrystalline cellulose, a disintegrating agent like corn starch or alginic acid, a lubricant such as magnesium stearate, a sweetening agent such as sucrose or lactose, or a flavoring agent. When a dosage form is a capsule, in addition to the above materials it may also contain a liquid carrier such as water, saline, a fatty oil. Other materials of various types may be used as coatings or as modifiers of the physical form of the dosage unit. Sterile compositions for injection can be formulated according to conventional pharmaceutical practice. Buffers, preservatives, antioxidants and the like can be incorporated according to accepted pharmaceutical practice.
In practicing the methods of this invention, the compounds of this invention may be used alone or in combination, or in combination with other therapeutic or diagnostic agents. In certain preferred embodiments, the compounds of this inventions may be coadministered along with other compounds typically prescribed for these conditions according to generally accepted medical practice, such as
The compounds of this invention can be utilized in vivo, ordinarily in mammals such as non-human primates, humans, sheep, horses, cattle, pigs, dogs, cats, rats and mice, or in vitro.